Ribosome biogenesis is essential for eukaryotic cells to progress through the cell cycle, grow in size, and divide. This application focuses on the processing of ribosomal RNA (rRNA), and the goal is to identify and study the function of critical factors involved in the cleavage of the pre-rRNAs that lead to the formation of the 18S rRNA. Using the yeast S. cerevisiae as a model system, the proposed work will focus on the function of the U3 small nucleolar ribonucleoprotein (snoRNP) and its subunits in these cleavage steps. The proposed research is important because the U3 snoRNP is ubiquitous in eukaryotes and is required for processing of the nascent rRNA transcript in all organisms studied so far. In yeast, the three U3 snoRNPdependent cleavage steps that lead to the release of the 1 8S rRNA occur via two different U3 snoRNA:pre-rRNA base-pairing interactions. Little is known about the function of the protein components of the U3 snoRNP in these RNA-RNA interactions or if protein-protein and protein-RNA interactions are essential for function. Many of the predicted U3 snoRNP protein components have not yet been identified. Preliminary results from the investigator's laboratory have led to the discovery of several novel U3 snoRNP components: MpplOp, Imp3p, and Imp4p. Their characterization has provided substantial evidence pointing to the critical nature of protein-protein, protein-RNA, and RNA-RNA interactions in snoRNP function. Aims 2, 3, and 4 will test specific hypotheses regarding these interactions and will examine the importance of each interaction in prerRNA processing. This will be accomplished by varied genetic approaches, including mutational analysis of both protein and RNA components. In Aim 1, the investigator will continue her promising initial efforts to purify the U3 snoRNP and to identify the remaining unknown protein components using affinity capture followed by mass spectrometry. In Aim 5, they propose to take advantage of a number of conditional alleles of MPP1O, JMP3, and IMP4 that they have already generated to set up a selected series of genetic screens. These screens are designed to identify new factors that function in the same pathway for pre-rRNA processing as the U3 snoRNP or regulate it in some manner. Taken together, this work will lead to a comprehensive understanding of the mechanisms underlying these essential U3 snoRNP-mediated RNA processing events.